Immunotherapy infusing antibodies, vaccines, and cells is an effective treatment approach for human malignancies. Potent anti-tumor effects are realized by harnessing the specificity and associated effector functions of the immune system to recognize and eliminate cancer cells. Clinical trials have revealed the advantages of immune-based therapies, including (i) defined mechanisms of action, (ii) defined specificity and reduced deleterious off-target effects, (iii) lower toxicities than conventional approaches. Adoptive cell therapy (ACT), based on the adoptive transfer of T cells genetically modified to enforce expression of a chimeric antigen receptor (CAR), has shown considerable promise in clinical trials treating tumors refractory to all other treatment methods. In particular, the use of CAR+ T cells rendered specific for CD19 demonstrated significant anti-tumor effects in patients with CD19+ chronic lymphocytic leukemia (CLL) refractory to conventional therapies. These trials infuse a heterogeneous population of genetically modified T cells which have been propagated to clinically-sufficient numbers. While the therapeutic potential of infused T cells depends on their persistence, immunocorrelative studies evaluating survival of infused T cells are currently limited to (i) describing the immunophenotypes and function of whole populations of the T-cell inoculum and (ii) immunophenotypic analyses on T cells recovered after infusion. This grant application seeks to adapt single- cell imaging and retrieval to inform on the potency of clinical-grade CD19-specific CAR+ T cells by developing a unified platform for assessing both phenotype and function on the few T cells (and tumor cells) directly obtained (without in vitro manipulation) from recipients of immunotherapy. Our objective is to use high throughput single-cell nanowell screening (SNS) that we have developed to undertake an in-depth quantitative functional characterization (multiplexed cytokine secretion, phenotype, cytotoxicity, effect of regulatory T cells, Treg) of pre-infusion CAR+ T cells and compare these data to T cells recovered from the patient, post-infusion. These data will be used to (i) quantify the functionalit of infused T cells and the potential for anti-tumor effects and (ii) improve the generation of T cells for greater efficacy in next-generation clinical trials. We will validate our approach in Specific Aim 1 where we will define the integrated functional and molecular profiles of clinical- grade CD19-specific CAR+ T cells. In Specific Aim 2 we will implement our methodology to quantify the in vivo persistence of adoptively transferred cells. This will test the hypothesis tha SNS can quantify the therapeutic potential of clinical-grade T cells and in the formation of next-generation clinical trials. PUBLIC HEALTH RELEVANCE: Genetically modified T cells are being infused in clinical trials to target leukemia and lymphomas. This grant application validates tools for investigating the ability of the infused T cells to target tumor cells and applies this approach to improve our understanding of their therapeutic benefit in clinical trials. This will enable investigators to develop approaches to generating even more effective T cells to target hematologic malignancies in next-generation clinical trials.